Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals.

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity. We next show that the locomotor response to d-amphetamine is enhanced in GlyRα2 knockout animals, and that this increase correlates with c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the neuronal ensemble size in the striatum in response to D-amphetamine in GlyRα2 KO mice. Finally, we show enhanced appetitive conditioning in GlyRα2 KO animals that is likely due to increased motivation, but not changes in associative learning or hedonic response. Taken together, we show that GlyRα2 is an important regulator of dopamine-stimulated striatal activity and function.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

Pharmacokinetic and pharmacodynamic considerations for treating sarcoptic mange with cross-relevance to Australian wildlife.

Sarcoptes scabiei is the microscopic burrowing mite responsible for sarcoptic mange, which is reported in approximately 150 mammalian species. In Australia, sarcoptic mange affects a number of native and introduced wildlife species, is particularly severe in bare-nosed wombats (Vombatus ursinus) and an emerging issue in koala and quenda. There are a variety of acaricides available for the treatment of sarcoptic mange which are generally effective in eliminating mites from humans and animals in captivity. In wild populations, effective treatment is challenging, and concerns exist regarding safety, efficacy and the potential emergence of acaricide resistance. There are risks where acaricides are used intensively or inadequately, which could adversely affect treatment success rates as well as animal welfare. While reviews on epidemiology, treatment strategies, and pathogenesis of sarcoptic mange in wildlife are available, there is currently no review evaluating the use of specific acaricides in the context of their pharmacokinetic and pharmacodynamic properties, and subsequent likelihood of emerging drug resistance, particularly for Australian wildlife. This review critically evaluates acaricides that have been utilised to treat sarcoptic mange in wildlife, including dosage forms and routes, pharmacokinetics, mode of action and efficacy. We also highlight the reports of resistance of S. scabiei to acaricides, including clinical and in vitro observations.

Startle Disease: New Molecular Insights into an Old Neurological Disorder.

Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback inhibition in the spinal cord and brainstem of affected individuals. Mutations within the glycine receptor (GlyR) subunit and glycine transporter 2 (GlyT2) genes have been identified in individuals with SD. Impaired inhibitory neurotransmission in SD is due to pre- and/or postsynaptic GlyR or presynaptic GlyT2 dysfunctions. Previous research has focused on mutated GlyRs and GlyT2 that impair ion channel/transporter function or trafficking. With insights provided by recently solved cryo-electron microscopy and X-ray structures of GlyRs, a detailed picture of structural transitions important for receptor gating has emerged, allowing a deeper understanding of SD at the molecular level. Moreover, studies on novel SD mutations have demonstrated a higher complexity of SD, with identification of additional clinical signs and symptoms and interaction partners representing key players for fine-tuning synaptic processes. Although our knowledge has steadily improved during the last years, changes in synaptic localization and GlyR or GlyT2 homeostasis under disease conditions are not yet completely understood. Combined proteomics, interactomics, and high-resolution microscopy techniques are required to reveal alterations in receptor dynamics at the synaptic level under disease conditions.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

MED27, SLC6A7, and MPPE1 Variants in a Complex Neurodevelopmental Disorder with Severe Dystonia.

BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Loss, Gain and Altered Function of GlyR α2 Subunit Mutations in Neurodevelopmental Disorders.

Glycine receptors (GlyRs) containing the α2 subunit govern cell fate, neuronal migration and synaptogenesis in the developing cortex and spinal cord. Rare missense variants and microdeletions in the X-linked GlyR α2 subunit gene (GLRA2) have been associated with human autism spectrum disorder (ASD), where they typically cause a loss-of-function via protein truncation, reduced cell-surface trafficking and/or reduced glycine sensitivity (e.g., GLRA2Δex8-9 and extracellular domain variants p.N109S and p.R126Q). However, the GlyR α2 missense variant p.R323L in the intracellular M3-M4 domain results in a gain-of-function characterized by slower synaptic decay times, longer duration active periods and increases in channel conductance. This study reports the functional characterization of four missense variants in GLRA2 associated with ASD or developmental disorders (p.V-22L, p.N38K, p.K213E, p.T269M) using a combination of bioinformatics, molecular dynamics simulations, cellular models of GlyR trafficking and electrophysiology in artificial synapses. The GlyR α2V-22L variant resulted in altered predicted signal peptide cleavage and a reduction in cell-surface expression, suggestive of a partial loss-of-function. Similarly, GlyR α2N38K homomers showed reduced cell-surface expression, a reduced affinity for glycine and a reduced magnitude of IPSCs in artificial synapses. By contrast, GlyR α2K213E homomers showed a slight reduction in cell-surface expression, but IPSCs were larger, with faster rise/decay times, suggesting a gain-of-function. Lastly, GlyR α2T269M homomers exhibited a high glycine sensitivity accompanied by a substantial leak current, suggestive of an altered function that could dramatically enhance glycinergic signaling. These results may explain the heterogeneity of clinical phenotypes associated with GLRA2 mutations and reveal that missense variants can result in a loss, gain or alteration of GlyR α2 function. In turn, these GlyR α2 missense variants are likely to either negatively or positively deregulate cortical progenitor homeostasis and neuronal migration in the developing brain, leading to changes in cognition, learning, and memory.

Clinical, genetic, and functional characterization of the glycine receptor ß-subunit A455P variant in a family affected by hyperekplexia syndrome.

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle responses affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced nonepileptic seizures. The genetic causes of the disease can vary, and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR ß-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to WT cells. Western blot analysis also revealed a reduced amount of GlyR ß protein both in cell lysates and isolated membrane fractions. In line with the above observations, coimmunoprecipitation assays suggested that the GlyR α1-subunit retained coassembly with ßA455P to form membrane-bound heteromeric receptors. Finally, structural modeling showed that the A455P mutation affected the interaction between the GlyR ß-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in the affected individual.

Clueless/CLUH regulates mitochondrial fission by promoting recruitment of Drp1 to mitochondria.

Mitochondrial fission is critically important for controlling mitochondrial morphology, function, quality and transport. Drp1 is the master regulator driving mitochondrial fission, but exactly how Drp1 is regulated remains unclear. Here, we identified Drosophila Clueless and its mammalian orthologue CLUH as key regulators of Drp1. As with loss of drp1, depletion of clueless or CLUH results in mitochondrial elongation, while as with drp1 overexpression, clueless or CLUH overexpression leads to mitochondrial fragmentation. Importantly, drp1 overexpression rescues adult lethality, tissue disintegration and mitochondrial defects of clueless null mutants in Drosophila. Mechanistically, Clueless and CLUH promote recruitment of Drp1 to mitochondria from the cytosol. This involves CLUH binding to mRNAs encoding Drp1 receptors MiD49 and Mff, and regulation of their translation. Our findings identify a crucial role of Clueless and CLUH in controlling mitochondrial fission through regulation of Drp1.

Drug dose and animal welfare: important considerations in the treatment of wildlife.

A recent publication in Parasitology Research by (Old et al. Parasitol Res 120:1077-1090, 2021) raises the topical and often controversial issue of the treatment of wildlife by personnel with little or no formal scientific training (e.g. wildlife carers). In a valuable contribution to the subject, Old and colleagues document a wide range of topical (pour-on) application doses and frequencies of moxidectin (Cydectin®) administered in situ to bare-nosed wombats (Vombatus ursinus) by members of the wildlife carer/treater community in southeast Australia to treat sarcoptic mange disease. This treatment occurred under minor use permits issued by the Australian Pesticides and Veterinary Management Authority (APVMA). These permits do not require veterinary supervision, although carers are registered and are expected to comply with the guidelines of this permit.The prevalence and severity of sarcoptic mange in wildlife is influenced by a variety of factors including mite biology, environmental conditions, population density, animal behaviour and immune susceptibility (Browne et al. Bioscience, 2021). In bare-nosed wombats, combinations of these elements play a substantial role in making the treatment of an already difficult disease more complex. (Moroni et al. Parasit Vectors 13:471, 2020) comment that any pharmacological treatment of free-ranging wildlife must consider these factors when assessing their feasibility and implications, especially in the context of emerging drug resistance and potential long-term ecological impacts. As individuals with significant interest in sarcoptic mange and representing a range of professional research and veterinary expertise, we see value in providing expert commentary on this issue.

Positive Allosteric Modulators of Glycine Receptors and Their Potential Use in Pain Therapies.

Glycine receptors are ligand-gated ion channels that mediate synaptic inhibition throughout the mammalian spinal cord, brainstem, and higher brain regions. They have recently emerged as promising targets for novel pain therapies due to their ability to produce antinociception by inhibiting nociceptive signals within the dorsal horn of the spinal cord. This has greatly enhanced the interest in developing positive allosteric modulators of glycine receptors. Several pharmaceutical companies and research facilities have attempted to identify new therapeutic leads by conducting large-scale screens of compound libraries, screening new derivatives from natural sources, or synthesizing novel compounds that mimic endogenous compounds with antinociceptive activity. Advances in structural techniques have also led to the publication of multiple high-resolution structures of the receptor, highlighting novel allosteric binding sites and providing additional information for previously identified binding sites. This has greatly enhanced our understanding of the functional properties of glycine receptors and expanded the structure activity relationships of novel pharmacophores. Despite this, glycine receptors are yet to be used as drug targets due to the difficulties in obtaining potent, selective modulators with favorable pharmacokinetic profiles that are devoid of side effects. This review presents a summary of the structural basis for how current compounds cause positive allosteric modulation of glycine receptors and discusses their therapeutic potential as analgesics. SIGNIFICANCE STATEMENT: Chronic pain is a major cause of disability, and in Western societies, this will only increase as the population ages. Despite the high level of prevalence and enormous socioeconomic burden incurred, treatment of chronic pain remains limited as it is often refractory to current analgesics, such as opioids. The National Institute for Drug Abuse has set finding effective, safe, nonaddictive strategies to manage chronic pain as their top priority. Positive allosteric modulators of glycine receptors may provide a therapeutic option.

Contribution of GlyR α3 Subunits to the Sensitivity and Effect of Ethanol in the Nucleus Accumbens.

The glycine receptor (GlyR), a ligand-gated ion channel, is critical for inhibitory neurotransmission in brainstem, spinal cord, and in supraspinal regions. Recent data from several laboratories have shown that GlyRs are expressed in the brain reward circuitry and that α1 and α2 are the principal subunits expressed in the nucleus accumbens (nAc). In the present study, we studied the sensitivity to ethanol of homomeric and heteromeric α3 GlyR subunits in HEK293 cells and dissociated neurons from the nAc. Finally, we explored ethanol-related behaviors in a Glra3 knockout mouse (Glra3 -/-). Studies in HEK293 cells showed that while homomeric α3 GlyR subunits were insensitive to ethanol, heteromeric α3ß GlyR subunits showed higher sensitivity to ethanol. Additionally, using electrophysiological recordings in dissociated accumbal neurons, we found that the glycine current density increased in Glra3 -/- mice and the GlyRs were less affected by ethanol and picrotoxin. We also examined the effect of ethanol on sedation and drinking behavior in Glra3 -/- mice and found that the duration in the loss of righting reflex (LORR) was unchanged compared to wild-type (WT) mice. On the other hand, using the drinking in the dark (DID) paradigm, we found that Glra3 -/- mice have a larger ethanol consumption compared to WT mice, and that this was already high during the first days of exposure to ethanol. Our results support the conclusion that heteromeric α3ß, but not homomeric α3, GlyRs are potentiated by ethanol. Also, the increase in GlyR and GABA A R mediated current densities in accumbal neurons in the KO mice support the presence of compensatory changes to α3 knock out. The increase in ethanol drinking in the Glra3 -/- mice might be associated to the reduction in ß and compensatory changes in other subunits in the receptor arrangement.

Glycine Transporters and Receptors as Targets for Analgesics.

The suitability of modulating glycinergic neurotransmission for the treatment of inflammatory and chronic pain has gained widespread recognition, with glycine receptors (GlyRs) and glycine transporters (GlyT1 and GlyT2) now considered key therapeutic targets [...].

Novel Functional Properties of Missense Mutations in the Glycine Receptor ß Subunit in Startle Disease.

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and ß subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR ß subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR ß variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR ß subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and ß subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR ß subunits to gephyrin aggregates. However, a significantly lower number of GlyR ß aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR ß subunit aggregates was increased in comparison to wild-type ß. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR ß-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR ß M3-M4 loop, we suggest that structural changes within the GlyR ß subunit result in differences in GlyR ß-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice.

BACKGROUND AND PURPOSE: Glycine receptors composed of α1 and ß subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol. EXPERIMENTAL APPROACH: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice. KEY RESULTS: Similar levels of α and ß receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αß receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol. CONCLUSION AND IMPLICATIONS: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.

Application of the random forest algorithm to Streptococcus pyogenes response regulator allele variation: from machine learning to evolutionary models.

Group A Streptococcus (GAS) is a globally significant bacterial pathogen. The GAS genotyping gold standard characterises the nucleotide variation of emm, which encodes a surface-exposed protein that is recombinogenic and under immune-based selection pressure. Within a supervised learning methodology, we tested three random forest (RF) algorithms (Guided, Ordinary, and Regularized) and 53 GAS response regulator (RR) allele types to infer six genomic traits (emm-type, emm-subtype, tissue and country of sample, clinical outcomes, and isolate invasiveness). The Guided, Ordinary, and Regularized RF classifiers inferred the emm-type with accuracies of 96.7%, 95.7%, and 95.2%, using ten, three, and four RR alleles in the feature set, respectively. Notably, we inferred the emm-type with 93.7% accuracy using only mga2 and lrp. We demonstrated a utility for inferring emm-subtype (89.9%), country (88.6%), invasiveness (84.7%), but not clinical (56.9%), or tissue (56.4%), which is consistent with the complexity of GAS pathophysiology. We identified a novel cell wall-spanning domain (SF5), and proposed evolutionary pathways depicting the 'contrariwise' and 'likewise' chimeric deletion-fusion of emm and enn. We identified an intermediate strain, which provides evidence of the time-dependent excision of mga regulon genes. Overall, our workflow advances the understanding of the GAS mga regulon and its plasticity.